The broad, long-term objective of this proposal is to elucidate the molecular mechanisms by which molecular chaperones promote heme signaling in eukaryotes. The molecular chaperones HSP90 and HSP70 play key roles in the proper functioning of signal transducers involved in many fundamental biological processes, such as steroid and heme signaling and cell cycle control. Importantly, my lab recently identified the heme activator protein Hap1 as a natural substrate of HSP90 and HSP70 in yeast. The Hap1 system thus provides a unique tool for exploring the bonafide functions of molecular chaperones in signal transduction by permitting the use of powerful yeast genetic and biochemical techniques. Hap1 is the master transcriptional regulator mediating the effects of oxygen on gene expression in yeast. Its activity is precisely controlled by heme. Hap1 has served as an excellent paradigm for studying heme signaling in eukaryotes. Here we will exploit the Hap1-heme regulatory system to investigate the roles of molecular chaperones in signal transduction. We propose to rigorously test the following hypotheses: (1) HSP90 and HSP70 directly interact with Hap1. (2) HSP90 promotes Hap1 activation by heme. (3) HSP70 and its cohorts mediate Hap1 repression in the absence of heme. Vigorous genetic and biochemical approaches will be applied to achieve three Specific Aims: (1) to dissect the molecular interactions of HSP90 and HSP70 with Hap1, (2) to determine the molecular mechanism by which HSP90 promotes Hap1 activation by heme, and (3) to determine the molecular mechanism by which Hap1 is repressed in the absence of heme. These studies should uncover novel mechanisms governing the actions of molecular chaperones in signal transduction and should provide a foundation for studying the actions of molecular chaperones in heme signaling in higher eukaryotes.